Trade-offs constrain adaptive pathways to the type VI secretion system survival

Summary The Type VI Secretion System (T6SS) is a nano-harpoon used by many bacteria to inject toxins into neighboring cells. While much is understood about mechanisms of T6SS-mediated toxicity, less is known about the ways that competitors can defend themselves against this attack, especially in the absence of their own T6SS. Here we subjected eight replicate populations of Escherichia coli to T6SS attack by Vibrio cholerae. Over ∼500 generations of competition, isolates of the E. coli populations evolved to survive T6SS attack an average of 27-fold better, through two convergently evolved pathways: apaH was mutated in six of the eight replicate populations, while the other two populations each had mutations in both yejM and yjeP. However, the mutations we identified are pleiotropic, reducing cellular growth rates, and increasing susceptibility to antibiotics and elevated pH. These trade-offs help us understand how the T6SS shapes the evolution of bacterial interactions.


Highlights
E. coli evolved 27-fold higher resistance to V. cholerae T6SS after 500 generations Convergent evolution reveals just mutational pathways for improving T6SS survival A mechanosensitive channel gene mutation provides broad spectrum resistance to T6SS Mutations improving T6SS survival reduce growth, revealing an evolutionary tradeoff

INTRODUCTION
Bacteria are one of the most common forms of life on Earth and often live in polymicrobial biofilms.Within this complex community, negative bacterial interactions are common, 1 due to intense competition for resources such as nutrients and space.One way for bacteria to gain an advantage over their competitors is by killing them.They have evolved two major classes of antagonistic mechanisms to eliminate competitors: diffusible and contact-dependent.4][5] On the other hand, contact-dependent antagonisms are less diverse and their social dynamics remain relatively understudied. 6One noteworthy counterexample is the Type VI secretion system (T6SS), discovered in 2006.The T6SS is a contact-dependent ''nano-harpoon'' that kills neighboring cells by injecting them with a set of toxic proteins. 76][17][18] Similar to antibiotic resistance, one strategy is to neutralize the toxins.0][21] However, cells lacking immunity proteins are vulnerable to the toxins.3][24][25] P. aeruginosa, a model organism for T6SS research, has evolved particularly sophisticated regulation of its T6SS.Cells that have been provoked by the T6SS of another bacterium can assemble their own T6SS apparatus and launch a counterattack in the direction from which the first attack came. 26P. aeruginosa is additionally able to induce T6SS attack in response to kin cell lysis, via a mechanism called ''danger sensing.'' 27Physical processes can also offer protection.9][30][31][32] External signaling can play a role in this protection, with recent reports that the presence of glucose enhances survival of E. coli cells to T6SS attack, mediated through cyclic AMP and its cognate target, the CRP regulator. 33Other regulators that coordinate stress response systems, such as Rcs and BaeSR may also play an important role, as deletions of these genes reduce survival from attack. 34,35

Experimental evolution of the type VI secretion system resistance
We report the development of an experimental evolution platform with two model organisms, to identify mechanisms by which bacteria can become resistant to T6SS attack (Figure 1A).2][43] The two species were co-cultured on agar plates in 10:1 ratio (killer:target) to ensure direct contact between cells, which is . Experimental evolution of resistance to V. cholerae's Type VI Secretion System (A) Experimental design.We experimentally evolved eight replicate populations of E. coli.Each round of selection included $16 generations of growth in liquid media, followed by co-culture with T6SS-expressing V. cholerae on solid media, where initially the vast majority of E. coli were killed.V. cholerae were removed via antibiotics, and the surviving E. coli resumed growth in liquid media.(B) Over 30 rounds of selection, E. coli in the T6SS treatment evolved a 27-fold increase in T6SS survival relative to the ancestral E. coli strain, while controls competed against a T6SS(À) V. cholerae did not evolve a significant increase in T6SS resistance.**** denotes a difference in survival with p % 0.0001, determined via ANOVA and a pre-planned contrast.
(C) Convergent evolution of genes affording T6SS survival.Three genes were mutated in all eight independently evolving populations: apaH arose in six, while mutations in yejM and yjeP arose in the other two populations.For deletions (D), numbers in parentheses refer to the nt position of the deletion.(8nt) 2->3 * refers to an 8 nt repeat that expanded from 2 repeats to 3 repeats long, resulting in a frameshift mutation.W249X refers to a premature stop codon at position 249, resulting in a protein product truncated near the C terminus.(AA = amino acids; nt = nucleotides).
necessary for T6SS attack.99.99% of our E. coli ancestor were killed by V. cholerae during the solid-media killing phase of the experiment, imposing strong selection for T6SS survival.Between rounds of competition, E. coli populations were grown for $16 generations in LB medium overnight, in the presence of kanamycin and chloramphenicol to prevent V. cholerae growth.We also evolved four control populations, competing the same ancestral E. coli against a T6SS-deficient V. cholerae DvasK strain.We reasoned that mutations arising in these four control populations would account for adaptation in our environment, including growth, dilution, and co-culture with V. cholerae on solid media, but not from injury from the T6SS.After 30 rounds of selection, evolved strains were an average of $27-fold more resistant to V. cholerae's T6SS attack, and the control populations had on average 3.9% higher survival, a negligible difference (F 11,71 = 15.8, p % 0.0001, ANOVA with replicate nested in treatment.Fold survival was log-transformed prior to analysis to homogenize variances, and treatment effect was assessed with pre-planned contrast, F 1,60 = 234, p % 0.0001; Figure 1B).

Identifying and characterizing key mutations
We identified mutations arising in our experiment by sequencing a single genotype from each population after 30 rounds of selection.With an average of 2.75 (standard deviation 1.09) mutations per genome in the experimentally evolved isolates of each population, we chose to focus on mutations that occurred in more than one replicate population, as convergent evolution strongly suggests these mutations are adaptive (Figure 1C; Figure S1).Six of the eight isolates had mutations in apaH.Four of which are frameshift mutations, suggesting they resulted in lossof-function (Figure 1C).This gene is responsible for the ''de-capping'' of mRNAs in a bacterial cell. 44Little is known about the global regulatory effect of loss of apaH, but it is hypothesized that a null mutation leads to RNA stabilization.Notably, the isolate from population E8 only gained a $3-fold increase in survival relative to its ancestor; which was significantly lower than five of the seven other replicate experimental populations (fold survival was log-transformed prior to analysis to homogenize variances, pairwise differences between each replicate population assessed via ANOVA and Tukey's HSD with overall significance at a = 0.05; Figure 1B).The mutation in apaH found in this isolate creates a premature stop codon near the end of the gene (amino acid 249 out of 280) that likely retains the partial function of apaH, resulting in a more modest survival advantage.Two of the eight isolates did not have a mutation in apaH.Instead, these two populations each had missense or frameshift mutations in both yjeP (also known as mscM) and yejM, suggesting an interaction between these two genes (Figure 1C).yjeP encodes a mechanosensitive channel that protects cells from osmotic shock. 45The gene yejM (also known as pbgA and lapC) encodes a metalloprotein that regulates bacterial lipopolysaccharides biosynthesis. 46,47Deletion of yejM is lethal in E. coli, while C-terminal truncation mutations result in the partial function of the gene. 48Both mutations we found in yejM occur near the C-terminus.
To test the function of mutations found in apaH, yjeP, and yejM independent of the role of other mutations that arose in experimental lineages (Figure S1), we re-engineered mutations in these genes in the ancestral strain.A clean deletion of apaH increases T6SS protection by 3-fold, whereas E. coli carrying a single copy of apaH expressed from a heterologous constitutive promoter at the Tn7 site is 0.4-fold more susceptible than the ancestor (fold survival was log-transformed prior to analysis to homogenize variances, comparison of means was assessed with one-sample t-test (m = 0) and Bonferroni correction with overall significance at a = 0.05, p % 0.0001 and p % 0.001; Figure 2A; see STAR Methods).yjeP I724T is a gain-of-function mutation that confers the type VI secretion system resistance yjeP is one of the four paralogs predicted to encode the MscS mechanosensory channel. 45An identical missense mutation in yjeP (yjeP I724T ) occurred independently in two lineages (Figure 1C), suggesting that this amino acid substitution enhances T6SS survival and represents a gain-of-function mutation.In the ancestor genetic background, we introduced a yjeP disruption, constitutively expressed yjeP, and reconstructed the yjeP I724T mutation.Interestingly, neither the absence of yjeP nor its constitutive expression affected T6SS survival.However, E. coli carrying the yjeP I724T mutation experienced a $4-fold survival benefit (fold survival was log-transformed prior to analysis to homogenize variances, pairwise differences between each replicate population assessed via ANOVA and Dunnett's test with overall significance at a = 0.05, p % 0.01; Figure 2B).
Because YjeP is predicted to be a mechanosensitive channel, 45 we determined how the yjeP I724T mutant responded to pH and osmotic shock, classic stressors for probing mechanosensor function.A yjeP null mutant (DyjeP) and a mutant expressing yjeP constitutitvely (yjeP*) behaved like WT Figure 3).Interestingly, while the yjeP I724T mutant was unaffected by changes in osmolarity (Figures 3A and 3B), it did exhibit highly significant decreases in maximum growth rate in the exponential phase when grown in alkaline conditions (Figures 3C and 3D), suggesting that YjeP may be an ion channel (OD 600 was log-transformed prior to analysis, and the growth rates were assessed with linear regression comparison of the slopes in the exponential phase with significance as detailed in the legend of Figure 3).To determine whether YjeP is the only MscS mechanosensitive channel protein that can affect T6SS resistance, we also tested one of the three YjeP homologs, YbdG, 45 because a prior study showed a ybdG I176T gain-of-function mutation also confers sensitivity to osmotic shock. 49Unlike yjeP I724T , the ybdG I167T did not confer T6SS resistance, nor did a ybdG null (Figure 3; Figure S7).Thus, we conclude that yjeP I724T is a gain-of-function, or co-dominant, mutation in an ion channel that confers T6SS resistance.

E. coli yjeP/yejM double mutants are much more resistant to diverse the type VI secretion system toxins
The fact that yejM and yjeP accrued mutations in parallel in two independent populations suggests there may be an epistatic relationship between these two mutations.To test this hypothesis, we introduced both yejM mutations into the ancestral E. coli without and with the yjeP I724T mutation.While the yjeP I724T mutation confers a modest benefit (4-fold increased survival; fold survival was log-transformed prior to analysis to homogenize variances, pairwise differences between each replicate population assessed via ANOVA and Dunnett's test with overall significance at a = 0.05, p % 0.01; Figure 2B), the presence of either yejM mutation by itself had no effect on resistance (Figure 2C).However, the yjeP I724T mutation combined with either yejM mutation enables a $40to 50-fold increase in survival compared to the ancestor (fold survival was log-transformed prior to analysis to homogenize variances, pairwise differences between each replicate population assessed via ANOVA and Dunnett's multiple comparison with overall significance at a = 0.05, p % 0.0001; Figure 2C).In other words, mutation in yejM increases resistance only in strains that also have the yjeP point mutation.
We next examined whether the mutations that arose in our experiment provide general resistance to T6SS attack, or are specific to the toxins employed by the V. cholerae C6706 strain, used in this evolution screen, which codes three auxiliary T6SS effectors in addition to the large cluster.We therefore competed each mutant E. coli strain against an environmental isolate of V. cholerae killer, BGT41 (also known as VC22), which encodes a constitutive T6SS with effectors predicted to have enzymatic activities distinct from those produced by C6706 and encountered by E. coli during experimental evolution. 50,51This environmental isolate is a superior killer of E. coli, relative to C6706, 51 necessitating that we perform our killing assays at a 1:4 killer:target ratio, rather than the 10:1 ratio used with C6706 (at the original 10:1 ratio, no E. coli survived).This change in ratio resulted in different values for fold-changes in survival compared to earlier plots.However, the relative survival increases of the yjeP I724T mutants rather than the value of the fold-increase, demonstrate general resistance.Evolved strains with yje-P I724T and yjeP/yejM double mutations survived significantly better than the E. coli ancestor, but apaH did not measurably increase survival (fold survival was log-transformed prior to analysis to homogenize variances, pairwise differences between each replicate population assessed via ANOVA and Dunnett's multiple comparison with overall significance at a = 0.05, p % 0.0001; Figure 4).In addition, unlike with the C6706 killer (Figure 2C), the yejM mutations did not further increase the survival of the yjeP I724T mutant (Figure 4).This suggests that I724T in yjeP may provide broad spectrum resistance to T6SS while protection conferred by mutations in the YejM C-terminus and in apaH may depend on the specific effector employed.

Two evolutionary pathways to the type VI secretion system resistance
Our results suggest two distinct mutational pathways lead to increased T6SS survival in E. coli.To test whether these pathways are independent of one another, or are instead mechanistically redundant, we constructed E. coli strains with the apaH disruption, yjeP I724T , and either yejM(8bp+) or yejM I427N .We refer to these strains as the ''triple mutants.''We compared each of the two triple mutants to their corresponding double mutants containing only yjeP I724T and one of the yejM C-terminal mutations.In other words, we sought to determine whether the deletion of apaH would further increase the resistance of the yejM/yjeP mutants, which survive the best in response to T6SS attack of our reconstructed strains.Both triple mutants survived T6SS attack an average of 7.5-and 4.5-fold better than their respective double mutants (or 111-and 66-fold better than the ancestor; Figure 5 correction with overall significance at a = 0.05, p % 0.0001, see STAR Methods) We therefore conclude that apaH disruption and yjeP/yejM mutation represent two mechanistically distinct evolutionary pathways to T6SS resistance.
Experimental evolution reveals a trade-off between the type VI secretion system resistance and growth rate So far we have shown that E. coli readily evolves resistance to T6SS attack, one of the most common mechanisms of antimicrobial warfare.Why, after billions of years of evolution, are bacteria still so poorly defended against the T6SS?Evolutionary theory predicts that tradeoffs between antibiotic resistance and other fitness-dependent traits can maintain susceptibility. 52To test this hypothesis, we examined the effect of each mutation on cellular growth rate by competing them against the ancestral genotype of E. coli, under the conditions that mirrored our selection experiment.Mutations in apaH, yejM, and yjeP decreased fitness during growth (Figure 6).In fact, there was an overall negative correlation between T6SS survival and growth rate for the strains generated in this study (log 10 (survival) = À2.9 log 10 (growth) -0.27, R 2 = 0.63, p = 2.34$10 À5 ; this regression excludes the crp and rlmE mutants (described later in discussion), as well as the triple mutants (green dots) that never arose during experimental evolution; Figure 6A).Our evolution experiment consisted of $16 generations of exponential growth in LB media each day, followed by a round of T6SS killing on plates.We thus calculated a fitness isocline across the phase space of this trade-off (black dashed line in Figure 6A), along which a mutant would have equal fitness to the ancestor across one round of growth and killing, with the equation y = 1=x (in log 10 space).For example, along this line, a 100-fold increase in T6SS survival is exactly canceled out by a 100-fold decrease in overnight growth.Mutations that lie above this line should be more fit than our ancestral strain, while mutations below the line should be maladaptive.Perhaps unsurprisingly, given the strong selection on both growth and T6SS survival, all mutations (and combinations) we identified in evolved isolates are adaptive.The triple mutants (yjeP/yjeM/apaH) suffer a growth defect strong enough to place them below the pink regression line where evolved isolates and reconstructed mutants lie.Because the triple mutants lie above the black fitness isocline, they are more fit in this fluctuating growth/attack cycle than the ancestor; however, they are less fit than the trend shown by the evolved isolates and other reconstructed mutants.This may explain why, despite a high degree of convergent evolution in our overall experiment, the combination of all three mutations was not seen in the sequenced isolates.
We also measured growth and survival rates for two mutants that did not arise in our experimentally evolved populations -strains with disruptive mutations in crp and rlmE.We have previously shown that the deletion of crp, a global transcriptional repressor, results in increased survival to the T6SS in E. coli, but also greatly reduces growth rate. 33,53,54While this mutation does fall above the fitness isocline, it did not appear in our evolution experiment (Figure 6).This could be because the decreased growth rate is too severe for the increase in T6SS resistance to be worth it.Because all of our mutations of interest result in decreased growth rate, we also sought to test whether decreased growth rate was sufficient to increase T6SS resistance.For example, slower growth could prevent microcolonies of the two strains from physical mutant has a significant decrease in division rate.Linked markers used to construct the mutants do not affect growth in the tested conditions (Figure S8).To determine if different genotypes grow at different rates as a function of pH, we examined log-transformed (with a constant of 1 added to each OD value prior to transformation to maintain positive values) OD over the first four hours of growth via ANCOVA.The relative growth rate of DyjeP::I724T, as measured by the interaction between Ln(OD) and strain, and confirmed by a Tukey's HSD post hoc test, was highly significant in alkaline conditions (pH 8.6), both when adjusted with KOH (F(4, 260) = 66.51, p < 0.0001) and NaOH (F(4, 260) = 35.22,p < 0.0001).In contrast, at pH 5.5 (F(4, 260) = 0.55, p = 0.7) and pH 7 (F(4, 260) = 1.53, p = 0.193), the interactions were not significant, suggesting that at these lower pHs all strains grew at the same rate.
contact on the plate during the course of the co-culture competition.We engineered a slow-growing strain of E. coli by deleting rlmE, which encodes for a methyltransferase that modifies the ribosomal RNA (rRNA) -an essential process for efficient protein synthesis.As a result of this deletion, the strain's growth was significantly hampered, amounting to just 0.14% of its unaltered ancestor's growth over a single cycle (twosided t-test, p = 8.75$10 À5 ).This strain results in much smaller colonies when growing on plates, but has only a 10-fold increase in survival when challenged with T6SS attack (Figure S9).The rlmE mutant is below the fitness isocline in Figure 6A, as the modest increase in survival is not commensurate with the massive growth defect of this mutant.This shows that slower growth is a side effect of mutations that increase T6SS resistance, not a cause of increased resistance.
Another trade-off we tested is susceptibility to aminoglycoside antibiotics.The apaH disruption strain has a significantly lower minimum inhibitory concentration (MIC) than the ancestor when grown in streptomycin and kanamycin (pairwise differences between each replicate population assessed via ANOVA and Dunnett's test with overall significance at a = 0.05, p % 0.0001 and p % 0.05; Figures 6B and 6C), meaning that they are more susceptible to these antibiotics.This is consistent with previous work on apaH. 55However, strains containing the yjeP point mutation did not show increased susceptibility.For our evolution experiment, we used E. coli with two antibiotic resistance genes (chloramphenicol and kanamycin), allowing us to use these antibiotics to remove all V. cholerae from the population each day after the killing phase of the experiment.Given that apaH disruptions result in increased kanamycin susceptibility, it is possible that the apaH and the kanamycin resistance genes share gene regulatory interactions.Inclusion of plasmid-encoded kanamycin resistance and kanamycin in the media may have eased the growth tradeoffs incurred by apaH mutations.

DISCUSSION
In this article, we use experimental evolution to examine how bacteria adapt to frequent T6SS exposure.We subjected populations of E. coli to alternating selection for rapid growth followed by attack by V. cholerae's T6SS (Figure 1A).All replicate populations evolving increased T6SS resistance (seven of the eight populations) utilized one of the two pathways: either a loss-of-function mutation in apaH; or a gain-offunction mutation I724T in yjeP combined with a partial loss-of-function in yejM, with both mutations necessary to provide a large survival advantage (Figures 1B and 1C).For a yjeP I724T mutant, the protection appears to be broad-spectrum, increasing resistance, by more than 3000-fold, to effector proteins not previously encountered in the experiment (Figure 4).Interestingly, the yjeP/yejM double mutants are also comparatively resistant to T6SS attack when competing against V. cholerae BGT41 (Figure 4), suggesting yjeP I724T provides a broader protection while the additional yejM mutations are specific to C6706 T6SS effectors (Figure 2C).While the mechanism underpinning this strain-specific effect is beyond the scope of this study, we hypothesize yejM I427N still encodes a partially functional YejM periplasmic domain, whereas an insertion of 8 nt (yejM1694) results in a frameshift mutation, resulting in a complete disruption of the C-terminus. 56In Salmonella enterica and E. coli, truncation of the C-terminus was shown to disrupt the function of YejM, negatively impacting lipopolysaccharide biosynthesis.This leads to a defective outer membrane that leaks periplasmic proteins into the extracellular space. 57Periplasmic leakage may reduce the concentration of membrane-localized T6SS toxins injected into E. coli bearing the yejM1694 mutation, reducing their lethality.In contrast, mutations in apaH were specific to the T6SS effectors they were evolved against, showing no efficacy against a different strain of V. cholerae with T6SS effectors previously not encountered (Figures 2A and 4).
Of the two primary mutational pathways we focused on in this study, it is interesting that the less beneficial path to T6SS resistance, loss of function in apaH, evolved more times than the far more beneficial combination of yjeP I724T and a partial loss-of-function of yejM (Figure 4).This is likely because it is easier to gain beneficial mutations in the apaH pathway: any loss of function mutation in the gene gives the phenotype, whereas the yejM/yjeP pathway requires more constrained mutations in two separate genes.The convergent evolution we observed in our experiment (identical yjeP SNPs in both populations evolving resistance via this mechanism) further suggests that specific mutations, not simple loss of function mutations, may be required in yjeP.Given the difference in T6SS resistance between evolved isolates with an apaH mutation (Figures 1B and 1C) and the constructed apaH mutant (Figure 2A), we hypothesize that other mutations acquired by the evolved populations may also contribute to T6SS survival.
Over 500 generations of experimental evolution in 8 replicate populations, we found just two pathways to increased T6SS resistance.The engineered triple mutants in both pathways confirm these two pathways are independent since both showed enhanced resistance relative to mutants in only a single pathway (Figure 5).While prior work has shown that many genes that can affect T6SS survival, 34,37,[58][59][60] implying that adaptation might be idiosyncratic among independent populations, our results suggest that adaptive routes to T6SS resistance are remarkably constrained.One possibility is that our populations are mutationally limited.This is unlikely, as we can expect $9.2 x 10 5 mutations to arise within each growth cycle (based on $10 10 cells being produced per cycle, a mutation rate of $0.2 x 10 À10 per generation per base 61 and a genome size of 4.6 MB), or 2.8 x 10 7 mutations in each population over the course of the experiment.Instead, the high degree of evolutionary convergence in our experiment suggests that there may simply be relatively few routes to increased T6SS survival in which the benefits of the mutation, integrated across the culture cycle to include pleiotropic costs, are great enough to drive the clonal lineage to high frequency.
3][64] While the details depend on taxon and environment, 64,65 antibiotic resistance often comes with trade-offs to other fitness components, [65][66][67][68] although this is not always observed. 69Trade-offs are often noted in regard to mutations in essential genes that are targeted by antibiotic drugs, such as genes encoding ribosomal proteins. 701][72][73] In contrast to diffusible antibiotics, the eco-evolutionary dynamics of contact-mediated killing remains less studied and it is unclear if or when similar compensatory adaptation would occur if we continued our experiment.The fact that we observe a strong trade-off between T6SS survival and growth rate is not entirely unexpected.The T6SS is an ancient, widespread, and highly effective microbial weapon.Trade-off free adaptations that increase survival to T6SS attack would be expected to rapidly fix in many bacterial populations.As a result, pleiotropic costs to T6SS resistance could play an important role in maintaining T6SS efficacy over evolutionary time.
Single mutations that confer resistance to an individual antibiotic are common, as a modification of one target site may be sufficient to escape drug toxicity. 74Because the probability a susceptible cell will simultaneously gain mutations allowing it to survive multiple antibiotics is far lower than the probability of gaining resistance to any single antibiotic, physiological mechanisms that afford broad-spectrum toxin resistance (e.g., efflux pumps) can often incur fitness trade-offs. 757][78][79] We thus might expect that, as in our experiment here, T6SS resistance often evolves via mechanisms that modify cellular physiology or behavior (e.g., increased capsule thickness) that improves survival, albeit with .Trade-offs between T6SS resistance and fitness during growth (A) Mutations conferring a larger T6SS survival advantage also resulted in a greater reduction to reproductive fitness.Plotted are the change in frequency of each mutant across one 16 generation growth assay, and one T6SS attack, following the protocols from our evolution experiment.The dashed gray line represents a fitness isocline, y = 1=x, where fitness across one complete round of growth and T6SS survival selection is equal to that of the ancestor.In other words, the isocline represents where increased fitness during T6SS survival is exactly outweighed by decreased fitness in the growth phase.The dashed pink line represents a simple linear regression highlighting the trade-off between increased survival and decreased growth rate over 500 generations of evolution, as well as strains engineered with key mutations; log 10 (survival) = À2.9 $log 10 (growth) -0.27, r 2 = 0.65, p = 3.01$10 À5 .This regression does not include the triple mutants, or the Dcrp and DrmlE mutants, which did not occur in our experiment.pleiotropic costs. 29In contrast to diffusible antibiotics, it may be more difficult for bacteria to evolve resistance to T6SS-delivered effectors.T6SS attacks synchronously deliver multiple effectors that target different components of the intoxicated cell, and delivery is direct, which minimizes dilution and dispersal of the toxins in a heterogeneous extracellular environment.The importance of social interactions in microbial ecology and evolution has been increasingly recognized in recent years. 80,81Antagonistic interactions appear to be more common than cooperation or commensalism, 1 at least for species that are capable of being cultured.The Type VI secretion system -a ballistic harpoon containing multiple types of toxins capable of quickly killing susceptible cells, represents the cutting-edge of microbial weaponry.In this article, we show that E. coli can indeed evolve substantial genetic resistance to T6SS assault, but doing so entails trade-offs with reproductive fitness.We also found that one convergently evolving solution appeared to provide effectorspecific protection, while the other appeared to be more general.So far, relatively little effort has gone into understanding the mechanisms (both genetic and behavioral) through which microbes can evolve to resist dying from T6SS antagonism -a crucial gap in our knowledge that limits our ability to understand the ecology and evolution of this widespread microbial weapon.Further work will be required to determine if trade-offs between T6SS survival and reproduction are found in other taxa, and whether such trade-offs can be mitigated over longer evolutionary timescales via compensatory mutation.

Limitations of the study
The current study is limited by examining T6SS resistance of E. coli to antagonism by an archetype V. cholerae strain (C6706) and one environmental strain.Future experiments will explore whether the principles established here also hold with C6706 carrying all possible combinations of its own four toxins, 2) additional V. cholerae killer strains that encode distinct toxins, 3) different T6SS Vibrio species, and 4) other genera of T6SS killers.This study also provides insights into mutations in genes that define evolutionary paths to T6SS resistance.Further studies characterizing these mutations in greater details will clarify the mechanisms by which each gene acts.

Bacterial and virus strains
Vibrio cholerae C6706 ptac-qstR Thomas, J.et al. 22 Killer strain V. cholerae C6706 ptac-qstR DvasK Thomas, J.et al. 22 Non-killer strain (T6-control) yjeJ and rlmE were deleted and replaced with the Amp R or Tet R cassette, respectively, by l Red recombination as previously described. 85o generate DyjeJ::ampR, the Amp R cassette from pUC19 was amplified by PCR using the primers KOyjeJBla.Fwd and KOyjeJBla.Rev, which contain homology to the 5 0 and 3 0 ends of yjeJ, respectively.To generate DrlmE::tetA, the tetA gene and promoter were amplified from Tn10 using the primers rrmJTET.Fwd and rrmJTET.Rev.DyjeJ::ampR or DrlmE::tetA DNA were transformed into DY378, a strain of E. coli K-12 that expresses the l Red recombination system from a temperature sensitive promoter.Prior to transformation, the l Red system was induced by incubating midlog phase DY378 cells at 42 C for 15 minutes in a shaking water bath.Recombinants were selected for on LB containing 25 mg mL À1 ampicillin (for DyjeJ::ampR) or 10 mg mL À1 tetracycline (for DrlmE::tetA).
To generate the DapaH::tetA, DybdG::tetA, and DyjeP::tetA null alleles, apaH::kanR, DybdG::kanR, and DyjeP::kanR from the Keio library 86 were moved into DY378 by P1vir transduction. 84The Kan R cassette in each Keio allele was replaced with tetA from Tn10 by l Red recombination. 85The tetA DNA was amplified by PCR using the primers pKD13TetA.Fwd and pKD13TetA.Rev, which contain homology to the 5 0 and 3 0 ends of the Kan R cassette, respectively.Recombinants were selected for on LB containing 10 mg mL À1 tetracycline and screened for sensitivity to 25 mg mL À1 kanamycin.
To generate the triple mutants, the tetracycline resistance cassette in DapaH::tetA was removed using the FLP/FRT system as previously described. 87The FRT-flanked araBAD::cat allele was reintroduced into the DapaH::frt strain by P1vir transduction.Mutations in yjeP and yejM were then introduced as described above.
ybdG I167T was constructed using CRISPR-Cas9 gene editing as previously described. 88The ybdG guide RNA plasmid pCRISPR-ybdG493 was constructed by ligating ybdG493.CRISPR duplexed DNA (Integrated DNA Technologies, IA, USA) into BsaI-digested pCRISPR.100 ng of pCRISPR-ybdG493 and 10 uM of the editing oligonucleotide ybdGI167T.MAGE (Integrated DNA Technologies, IA, USA) were transformed into MG3686, a derivative of DY378 that constitutively expresses Cas9 from a plasmid.Transformants were selected for on LB containing 25 mg mL À1 chloramphenicol and 50 mg mL À1 kanamycin.Recombinants containing the ybdG I167T mutation were identified by DNA sequencing (Azenta Life Sciences, MA, USA).Two phosphorothioate bonds were added at the 5 0 and 3 0 ends of the ybdGI167T.MAGE oligonucleotide to increase stability.
Genes were inserted at the Tn7 attachment site following a similar protocol described previously. 89,90Wildtype apaH or yjeP expressed from the constitutive promoter J23119 (http://parts.igem.org/Part:BBa_J23119)were cloned into XhoI and HindIII (New England Biolabs, MA, USA) digested pZS21, resulting in the plasmids pZS21-apaH and pZS21-yjeP.The J23119 promoter, gene, and rrnB1 terminator from pZS21-apaH or pZS21-yjeP were amplified by PCR using the primers pGRG25GA.Fwd and pGRG25GA.Rev.The U streptomycin/spectinomycin resistance cassette from pHP45U was amplified using the primers pGRG25SpcGA.Fwd and pGRG25SpcGA.Rev.apaH or yjeP DNA along with DNA corresponding to the U streptomycin/spectinomycin resistance cassette were inserted into PacI and AvrII digested pGRG25-Modular-BamA-Kan by Gibson Assembly (New England Biolabs, MA, USA).The resulting plasmids were transformed into MG1655 and transformants were selected for on LB containing 25 mg mL À1 spectinomycin and 0.2% (w/v) arabinose.Transformants were screened for integration of apaH or yjeP and the U streptomycin/spectinomycin resistance cassette at the Tn7 site by PCR.
V. cholerae was genetically engineered with established allelic exchange methods using vector pKAS32. 91Expression of chromosomal qstR from a heterologous Ptac promoter results in constitutive T6SS activity because C6706 lacks a functional lacI gene. 92An in-frame deletion of vasK prevents T6SS assembly. 22All Insertions, deletions, and mutations were confirmed by PCR and DNA sequencing (Eton Bioscience Inc, NC, USA).

Experimental evolution
Twelve replicate populations of E. coli with chromosomal Cm R cassette and a plasmid encoding Kan R cassette were initiated from an overnight culture in LB with kanamycin and chloramphenicol.Each day, cultures were washed twice with LB to remove antibiotics, then mixed with an overnight culture of either V. cholerae C6706 qstR* (for the 8 experimental populations) or C6706 qstR* DvasK (for the 4 control populations) in a 10:1 killer to target ratio.50 mL of each mixture was spotted onto an LB agar plate, dried, and incubated at 37 C for 3 hours.Competition mixtures were then resuspended in 5 mL of ddH 2 O containing kanamycin (50 mg mL À1 ) and chloramphenicol (10 mg mL À1 ), and put at 4 C for 30 minutes, conditions which allow for survival of E. coli but not V. cholerae.Surviving cells were then diluted 10-fold into LB containing kanamycin (50 mg mL À1 ) and chloramphenicol (10 mg mL À1 ) for overnight growth at 37 C.This procedure was repeated daily for 30 rounds.A sample of each whole population was frozen at À80 C with 25% glycerol every five days.The populations themselves were frozen at day 15 to briefly pause the evolution experiment, and were revived by diluting 500 mL thawed stock into 5 mL LB with kanamycin and chloramphenicol, and grown overnight to start the evolution again the next day.At the end of 30 rounds, a clonal isolate from each population was taken for subsequent phenotypic and genomic testing.

Stress assay
The optical density (OD 600 ) of overnight cultures of E. coli strains growing in the basal medium (pH 7) was measured by a ThermoFisher Scientific Genesys 30 spectrophotometer (MA, USA) and normalized to 1. Then cells were diluted 1:50 into the basal medium (pH 5.5, pH 7, and pH 8.6 with KOH or NaOH) in a 96-microtiter plate, which was incubated aerobically at 37 C with shaking in a BioTek Synergy H1 microplate reader (VT, USA).The OD 600 of each well was read every 30 mins for 16 h.A curve of best fit was assigned to each well using the 4P Growth model in JMP (JMPâ, Version 16.1.0.SAS Institute Inc., Cary, NC, 1989-2021), and the value of the ''Division'' parameter was compared across treatments and replicates.

Figure 2 .
Figure 2.While all mutations of interest increase T6SS resistance in various degrees, the yjeP/yejM double mutants survive significantly better (A) E. coli with the deletion of apaH or (B) yjeP I724T mutation had a slight increase in T6SS resistance relative to the ancestral E. coli strain, which was not observed in the other variants.(C) The combination of yjeP I724T and mutations in the C-terminus of YejM significantly improved the E. coli survival by more than 42-fold.Linked markers used to construct the mutants are not indicated in the figure.****, ***, and ** denote differences in survival with p % 0.0001, p % 0.001, and p % 0.01 respectively, determined via ANOVA and Dunnett's Multiple Comparison.

Figure 3 .
Figure3.E. coli yjeP I724T has reduced fitness under basic conditions E. coli and its yjeP derivatives grow similarly under acidic (A) and neutral pH.(B) In basic media adjusted with either KOH (C) or NaOH (D), however, the yjeP I724T mutant has a significant decrease in division rate.Linked markers used to construct the mutants do not affect growth in the tested conditions (FigureS8).To determine if different genotypes grow at different rates as a function of pH, we examined log-transformed (with a constant of 1 added to each OD value prior to transformation to maintain positive values) OD over the first four hours of growth via ANCOVA.The relative growth rate of DyjeP::I724T, as measured by the interaction between Ln(OD) and strain, and confirmed by a Tukey's HSD post hoc test, was highly significant in alkaline conditions (pH 8.6), both when adjusted with KOH (F(4, 260) = 66.51, p < 0.0001) and NaOH (F(4, 260) = 35.22,p < 0.0001).In contrast, at pH 5.5 (F(4, 260) = 0.55, p = 0.7) and pH 7 (F(4, 260) = 1.53, p = 0.193), the interactions were not significant, suggesting that at these lower pHs all strains grew at the same rate.

Figure 4 .
Figure 4.The yjeP and the yjeP/yejM mutants provide general resistance to T6SS attack When competed against V. cholerae with a set of toxins not encountered during experimental evolution, E. coli mutants with yjeP I724T had an increase in T6SS resistance relative the ancestral E. coli strain, whereas the deletion of apaH did not provide protection against T6SS effectors not encountered prior.Linked markers used to construct the mutants are not indicated in the figure.**** denotes a difference in survival with p % 0.0001, determined via ANOVA and Dunnett's Multiple Comparison.

Figure 5 .
Figure 5. Triple mutants are more resistant to T6SS attack than double mutants The addition of an apaH disruption further increases resistance of strains that already have the yjeP I724T /yejM C-terminal mutation genotype.Relative survival is shown for triple mutants with each type of yejM mutation.Values are shown as fold-increases relative to the corresponding double mutants lacking the apaH disruption.Linked markers used to construct the mutants are not indicated in the figure.**** denotes a difference in survival with p % 0.0001, comparison of means was assessed with ANOVA and Dunnett's Multiple Comparison (m = 0) with overall significance at a = 0.05.

Figure 6
Figure 6.Trade-offs between T6SS resistance and fitness during growth (A) Mutations conferring a larger T6SS survival advantage also resulted in a greater reduction to reproductive fitness.Plotted are the change in frequency of each mutant across one 16 generation growth assay, and one T6SS attack, following the protocols from our evolution experiment.The dashed gray line represents a fitness isocline, y = 1=x, where fitness across one complete round of growth and T6SS survival selection is equal to that of the ancestor.In other words, the isocline represents where increased fitness during T6SS survival is exactly outweighed by decreased fitness in the growth phase.The dashed pink line represents a simple linear regression highlighting the trade-off between increased survival and decreased growth rate over 500 generations of evolution, as well as strains engineered with key mutations; log 10 (survival) = À2.9 $log 10 (growth) -0.27, r 2 = 0.65, p = 3.01$10 À5 .This regression does not include the triple mutants, or the Dcrp and DrmlE mutants, which did not occur in our experiment.(B and C) Disruption of apaH results in decreased MIC for streptomycin (B) and kanamycin (C).The point mutation yjeP I724T does not affect susceptibility to these antibiotics.Linked markers used to construct the mutants are not indicated in the figure.**** and * denote differences in survival with p % 0.0001 and p % 0.05, determined via ANOVA and Dunnett's Multiple Comparison test.
Figure 6.Trade-offs between T6SS resistance and fitness during growth (A) Mutations conferring a larger T6SS survival advantage also resulted in a greater reduction to reproductive fitness.Plotted are the change in frequency of each mutant across one 16 generation growth assay, and one T6SS attack, following the protocols from our evolution experiment.The dashed gray line represents a fitness isocline, y = 1=x, where fitness across one complete round of growth and T6SS survival selection is equal to that of the ancestor.In other words, the isocline represents where increased fitness during T6SS survival is exactly outweighed by decreased fitness in the growth phase.The dashed pink line represents a simple linear regression highlighting the trade-off between increased survival and decreased growth rate over 500 generations of evolution, as well as strains engineered with key mutations; log 10 (survival) = À2.9 $log 10 (growth) -0.27, r 2 = 0.65, p = 3.01$10 À5 .This regression does not include the triple mutants, or the Dcrp and DrmlE mutants, which did not occur in our experiment.(B and C) Disruption of apaH results in decreased MIC for streptomycin (B) and kanamycin (C).The point mutation yjeP I724T does not affect susceptibility to these antibiotics.Linked markers used to construct the mutants are not indicated in the figure.**** and * denote differences in survival with p % 0.0001 and p % 0.05, determined via ANOVA and Dunnett's Multiple Comparison test.